Background: The goals of this randomized double-blind trial were to assess the antimicrobial activity in vivo of Sodium hypochlorite (NaOCl) vs. chlorhexidine gluconate (CHX) used in combination either with EndoActivator® or IRRI S® files in patients with apical periodontitis. Material and Methods: A total of 120 patients with apical periodontitis (in single or multiple root canals) were randomly assigned to the four irrigation protocols outlined below: Group A: 5.25% sodium hypochlorite (NaOCl) + EndoActivator®; Group B: 5.25% NaOCl + IRRI S® files; Group C: 2% chlorhexidine gluconate (CHX) + EndoActivator®; Group D: 2% CHX + IRRI S® files. Paper points were used to collect microbiological samples before (1A samples) and after (1B samples) irrigation. Viable colony-forming units (CFU) were quantified twice: (1) without speciation, and (2) only for Enterococcus Faecalis ( EF ). Statistical analysis was performed using SPSS 22.0 for Windows. Results: No significant differences were observed between NaOCl and CHX in the reduction of CFU; in fact, reduction was > 93% for the two irrigants. Conversely, statistically significant differences were found between the two activation techniques (sonic and ultrasonic) in the reduction of Enterococcus faecalis ( EF ). Thus, the effectiveness of ultrasonic activation was significantly higher (> 93%; p=0.012) as compared to sonic activation. Following the combination of the two irrigants with the two activation techniques (groups A, B, C and D), significant differences were observed between group A and B ( p =0.025) in the reduction of EF populations, reaching up to 94%. Conclusions: NaClO and CHX are effective in reducing intracanal bacterial load. Ultrasonic activation is the most effective activation technique in reducing EF populations.